Ethical statement
Prior to the experiment, the protocol was approved by the Ethics Committee of Valencia University, Spain (A1434714637496). The guidelines indicated by the Council Directive of the European Union (53/2013; February 1, 2013) for animal experimentation and the ethical rules proposed by Royal Decree 223, March 14 and October 13, 1988, were fulfilled. The study was reported following the ARRIVE guidelines.
Study design and experimental animals
Sixteen New Zealand rabbits of about 24 weeks of mean age and 3 to 4 kg of weight were used for the experiment. The animals were divided into two groups, eight animals per group, based on the period of euthanasia that was fixed to 1 and 8 weeks from the surgery.
Randomization and allocation concealment
The randomization was performed electronically (www.randomization.com) by an author that was not involved in the selection of the animals and in the surgical procedures (DB). The surgeon (JVA) was informed about the allocation treatment at the access window after having performed the sinus lift.
Sample size
Data from a study in rabbits [18] showed differences in the bone formation of about 10%. Considering this value as clinically relevant, and supposing a slightly greater variability (standard deviation ± 6%), a power of 0.8, and α = 0.05, a sample of 7 animals was considered sufficient to disclose differences. The number was then increased to 8 to compensate for the possible loss of animals.
Clinical procedures
Before surgery, a premedication was performed in all animals using 22 mg/kg of ketamine (Ketolar®; Pfizer, Madrid, Spain) and 2.5 mg/kg of xylazine (Rompun®, Bayer, Germany). As general anesthesia 1.5 mg/kg of propofol (Diprivan®, IL, USA) endovenous and maintained subsequently with isoflurane 2% and oxygen. During surgery, an intramuscular injection of 2.5 mg/kg of morphine 1%, (Braun®, Melsungen, Germany) was administered. As local anesthesia, 4% articaine with epinephrine 1/100.000 (ULTRACAIN DS forte®, Hoechst GmbH, Frankfurt, Germany) was used both in the tibia and in the nasal dorsum regions.
The skin at the proximal tibia was shaved and disinfected with Betadine (MEDA Pharma S.L., Madrid, Spain), and an incision was performed several millimeters below the anterior tibial tuberosity. The flaps were elevated to expose the tibial bone of the proximal region (Fig. 1a). Bone particles were collected using a Savescraper Twist curve (CGM spa, Divisione Medicale META, Reggio Emilia, Italy) and maintained in saline. The wounds were sutured with nylon (Aragó®, Barcelona, Spain).
Afterward, a trichotomy was performed in the nasal dorsum and, after disinfection of the experimental region using Betadine (MEDA Pharma®, Madrid, Spain), a sagittal incision was carried out. The skin and the periosteum were dissected and shifted laterally to expose the nasal bone. Antrostomies, 4 × 4 mm in dimensions, located about 3–4 mm laterally to the midline and about 10 mm in front of the nasal-frontal suture, were prepared bilaterally grinding the bone with a round diamond bur (Fig. 1b), under conspicuous irrigation with saline.
The sinus mucosa was elevated from the bone walls with a small elevator (Bontempi®, San Giovanni in Marignano, RN, Italy), and the space obtained was grafted with a collagenated cortico-cancellous porcine bone (OsteoBiol Gen-Os, Tecnoss®, Giaveno, Italy; 250–1000 μm). The autogenous bone particles were placed in the antrostomy and the subjacent region at the randomly assigned sites (treated sites; Fig. 1c, d). Collagen membranes (OsteoBiol® Evolution 0.3 mm, Tecnoss®, Giaveno, Italy; Fig. 1e) were placed to cover the antrostomies both at the treated and untreated sites. Resorbable sutures (Vicryl®, Johnson-Johnson, New Brunswick, NJ, USA) were used to close both periosteum and skin (Fig. 1f).
Maintenance care
Meloxicam (Normon®, Madrid, Spain) 0.2 mg/kg once a day for 7 days and Buprenorphine hydrochloride (Buprex®, Hull, UK) 0.02 mg/kg twice a day for 3 days were administered subcutaneously. The rabbits were kept in individual cages in controlled temperature rooms at the animal facilities of the University of Valencia. The monitoring of wounds and biological functions was performed for the whole follow-up.
Euthanasia
The animals were euthanized using 50 mg/kg of sodium pentobarbital (Nembutal® Schaumburg, IL, USA).
Paraffin sections preparation
The experimental region was dissected and fixed for 7 days at room temperature in formalin 10% and subsequently decalcified in Osteosoft (Merck KGaA®, Darmstadt, Germany). After decalcification, the specimens were washed in distilled water and included in paraffin. Sections of about 5 μm in width were prepared in a microtome (RM2245, Leica Biosystems, Wëtzlar, Germany). A section from the central region of the antrostomy was selected and stained with scarlet-acid fuchsine and toluidine blue and used for histomorphometric analysis.
Histomorphometric analysis
Overlapping calibrated digital images of the tissues were recorded with Leica Applications Suite version 4.4.0 software from a bright field Leica DM4000 B microscope (Leica Microsystems GmbH, Wëtzlar, Germany) equipped with a 5× lens and DFC420 digital camera. Single images were pasted and merged to compose each elevated sinus using the program Photoshop (Adobe Photoshop CC 2015.0.0).
The histomorphometric measurements were taken by an expert assessor (JM) using a lattice with squares of 75 μm. The following different areas were assessed within the grafted regions (Fig. 2): (i) close to the bone walls (Bone walls region), (ii) center of the augmented region (middle region), (iii) subjacent to the sinus mucosa (sub-mucosa region), (iv) and close to the antrostomy (close-to-window region). Moreover, the antrostomy (antrostomy region) was also evaluated in three different areas: adjacent to the medial and lateral edges, and the middle of the access window (Fig. 2). The tissues evaluated were newly formed bone, graft material, and soft tissues. Percentages were subsequently calculated.
Data analysis
The sites that received autogenous bone in the antrostomy and the subjacent regions (close-to-window) were considered as a test (treated sites) while the sites that did not receive the autogenous bone were considered as control (untreated sites).
The primary variable was the new bone percentage in the region of major interest that was the antrostomy and close-to-window areas. The mean new bone within the augmented sinus was considered as a secondary variable. Mean values and standard deviations were calculated for each outcome variable. The results were calculated using the software Excel 2013 (Microsoft Corporation, Redmond, WA, USA), while the statistical analyses were performed with the IBM SPSS Statistics software v.19 (IBM Inc., Chicago, IL, USA). In the text are reported only mean values and standard deviations while the tables were complemented with 25th, 50th (median), and 75th percentiles were also added. The Wilcoxon test was used to evaluate differences between the two sites with a level of significance set at 5%.